PROJECT 2 ABSTRACT The long-term objective of this Project centers on elucidation of the mechanisms whereby extra-cellular signals are sensed by the cell cycle machinery and regulate cell cycle progression during neoplastic conversion of epithelium. Our current work focus on the mitogenically responsive D-type cyclin, cyclin D1, and more specifically how the aberrant accumulation of cyclin D1 contributes to esophageal cancers and whether our enhanced understanding of cyclin D1 regulation can contribute to new and more effective therapies for this deadly disease. Progress in the characterization of the mechanism of cyclin D1 overexpression in cancer has been hindered by lack of information regarding the nature of the E3 ubiquitin ligase that directs cyclin D1 proteolysis. We have identified the SCF (FBX4-B crystallin) that controls cyclin D1 ubiquitination and degradation in esophageal cancer. Importantly, the progress we have made during the past 5 years has provided key insights into the mechanisms whereby dysregulated cyclin D/CDK4 activity contributes to the neoplastic conversion of a normal cell and to the ongoing proliferation and survival of esophageal tumor clones. This progress now provides a framework critical for proper utilization, evaluation and interpretation of the effects that can be achieved by treatment of cancer patients with the evolution of highly specific small molecule inhibitors of the cyclin D1/CDK4 kinase. Having developed this framework describing mechanisms of cyclin D1 dysregulation in esophageal cancer (in particular, esophageal squamous cell cancer or ESCC), we are now in an ideal position to translate these novel concepts and in doing so develop effective therapies for a cancer lacking in any significant therapeutic intervention. As direct result of this progress, we now propose an overarching new hypothesis wherein the SCFFBX4-B crystallin E3 ligase maintains threshold levels of the cyclin D1/CDK4 kinase critical for esophageal cell growth and homeostasis. We further hypothesize that therapeutic strategies targeting the cyclin D1/CDK4 kinase, or key downstream effectors (such as PRMT5), could be of significant therapeutic benefit in the treatment of esophageal cancer. Experiments proposed in this grant will directly determine the physiological contribution of Fbxo4 to the regulation of cyclin D1 accumulation and neoplastic activity in esophageal epithelium (Aim 1); assess the therapeutic efficacy of targeting the catalytic partner for cyclin D1 (CDK4/6) in esophageal cancer (Aim 2); and finally, address the mechanistic contributions of the histone methyltransferase, PRMT5, to the neoplastic activities of cyclin D1/CDK4 in esophageal cancer (Aim 3). Collectively, this unified approach is utilizing mechanistic findings as a platform for new translational/therapeutic strategies in ESCC that have potential applicability to other related cancers, such as head/neck squamous and lung squamous cell cancers. Our mechanistic approaches and translational applications are unified by integrated and synergistic approached with Projects 1 and 3, all supported by the Core Facilities.